Human amylin, a pancreatic peptide hormone, is the principal constituent of amyloid deposits in the pancreas, a hallmark of type-2 Diabetes Mellitus (T2DM). Aggregation of human amylin into small soluble oligomers and -sheet-enriched fibrils is linked to dysfunctions of beta ()- and other islet cells and pathogenesis of T2DM. Although much progress has been made in understanding the actions of amylin oligomers and aggregates, the cellular events and factors that contribute to amylin oligomerization, aggregation and toxicity in the pancreas remain largely unknown. The overarching goal of this proposal is to elucidate factors regulating amylin oligomerization and aggregation in the pancreatic human islet cells, and to determine the underlying mechanisms of amylin-mediated islet cell death. Our published and preliminary results suggest that plasma membrane (PM) cholesterol and endocytosis play a key role in amylin turnover and toxicity in the human islet cells. In the proposed studies, we will test the hypothesis that the amylin receptor (AM-R), clathrin and v- SNARE/Rab endocytotic proteins mediate amylin uptake and degradation in lysosomes, the impairment of which stimulates amylin oligomerization and aggregation, oxidative stress and consequently apoptosis of human islet cells. To test this idea, amylin trafficking, degradation and toxicity pathways in cultured human islet cells will be explored by combinations of atomic force microscopy (AFM), confocal microscopy, spectroscopy and biochemical approaches. The objectives of the proposal are to: (1) elucidate amylin trafficking and degradation pathways in human islet cells, and determine the extent to which they contribute to formation and accumulation of toxic amylin oligomers and aggregates, (2) determine the causal relationship between amylin turnover and toxicity in human islet cells, and (3) determine the mechanisms by which amylin oligomers and/or aggregates stimulate oxidative stress in human islet cells and thereby activate redox-sensitive stress kinases, apoptosis signal-regulating kinase 1 (ASK-1) and c-Jun N-terminal kinase (JNK). These studies should provide a better understanding of the molecular and biochemical events leading to formation of amyloid aggregates and soluble toxic amylin oligomers, knowledge which is critical for the development of novel therapeutics for the treatment of T2DM.